PROJECT ABSTRACT Proposal is designed in response to NHLBI/NIDA RFA-HL-19-028 (Sleep and Circadian-Dependent Mechanisms Contributing to Opioid Use Disorder (OUD) and Response to Medication Assisted Treatment (MAT)) as part of the HEAL Initiative. Annual costs attributed to the opioid crisis in the United States have shattered previous estimates. Synthetic opioids like fentanyl have surpassed prescription opioids as the leading cause of death from drug overdose and illicit fentanyl use continues to ride in regions with already high base rates of OUD. Among the most common symptoms experienced by individuals suffering with OUD are severe and persistent sleep and circadian disruptions. These sleep and circadian abnormalities are speculated to foster opioid craving and relapse, although direct evidence is limited. We lack a basic understanding of the mechanisms contributing to opioid dependence and their relationship to sleep and circadian systems. We identified a circadian-dependent mechanism which modulates fentanyl reward-related behaviors potentially via cell-type specific action in a key brain region linked to OUD?the nucleus accumbens (NAc). A majority of the neurons in the NAc are GABAergic medium spiny neurons which predominantly express either dopamine 1 or 2 receptors (D1R-MSNs or D2R-MSNs). These MSN subpopulations exert opposing and sometimes complementary actions on reward-related behaviors, whereby activation of D1R-MSNs drives drug seeking and relapse, while D2R-MSNs attenuate these behaviors. D1R-MSNs are also involved in the regulation of sleep-wake cycles. Our preliminary work suggests the diurnal excitability of NAc D1R-MSNs and their response to fentanyl is directly modulated by the circadian transcription factor NPAS2. We also show NPAS2 impacts sleep-wake cycles, potentially via action in NAc. Findings from human genetics studies indicate variants of NPAS2 are associated with alterations in the diurnal variation of anhedonia and motivation, suggesting links between NPAS2 and the circadian modulation of reward and motivation. We identified a novel role for NPAS2 to regulate fentanyl reward via activity in D1-MSNs of the NAc. We will use a combination of behavioral (i.e., self-administration and sleep polysomnography), slice electrophysiology, and molecular (i.e., cell-type specific RNA-seq) approaches to: Investigate the role of NPAS2 in D1R-MSNs to modulate the behavioral responses to fentanyl (Aim 1); Assess the impact of fentanyl on synaptic plasticity at D1R-MSNs and investigate whether NPAS2 mediates the potentiation of excitatory synapses at specific diurnal phases (Aim 2); Elucidate the cell-type specific NPAS2-dependent transcriptional mechanisms of fentanyl-seeking and relapse behaviors (Aim 3); and, Investigate whether NPAS2 rescue and buprenorphine MAT improve fentanyl-induced sleep disturbances (Aim 4). Our proposal will define the role for circadian-dependent transcriptional mechanisms in key reward circuits of opioid reward and uncover the therapeutic potential of NPAS2 for opioid dependence and relapse. Our studies will provide novel insights into the mechanisms at the intersection of sleep, circadian rhythms, and opioids.